Plasma C-Reactive Protein (CRP) level is a biomarker that predicts future risk of cardiovascular disease (CVD). We recently demonstrated that alleles at several common single nucleotide polymorphisms (SNPs) in the CRP gene correlate with plasma CRP levels, and functionally alter the regulation of the CRP promoter. However, the majority of the inter-individual variance in CRP levels remains unexplained. Common SNPs tend to be older than rare SNPs, and therefore have been exposed to longer term natural selection. Thus, it is not unlikely that rare SNPs exist with larger effects on plasma CRP than the known, common SNPs, and these rare SNPs might explain a significant fraction of the remaining variance in plasma CRP. The goal of this application is to discover rare SNPs in the CRP gene, which are of functional relevance in relation to plasma CRP levels and atherosclerotic pathogenesis. Aim 1: We will identify rare SNPs likely to alter CRP levels by resequencing two panels: A. the high (N=376) and low (N=376) tails of the CRP distribution (and age/gender matched controls) in the CARDIA cohort, a cohort of more than 4000 individuals, ages 38 to 50, and B. Severely stenosed cases (N=500) and controls (N=500) from a study of carotid atherosclerotic disease. Aim 2: We will functionally characterize putatively functional SNPs identified in Aim 1, using site-directed mutagenesis of a plasmid containing a CRP-GFP fusion gene to generate allelic constructs, and transient transfection analysis of expression from the allelic constructs to assess functional impacts on protein and RNA levels.